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The mesenchymal stem cell (MSC) secretome has been considered an innovative therapeutic biological approach, able to modulate cellular crosstalk and functionality for enhanced tissue repair and regeneration. This study aims to evaluate the functionality of the secretome isolated from periosteum-derived MSCs, from either basal or osteogenic-induced conditions, in the healing of a critical size calvarial bone defect in the rabbit model. A bioceramic xenograft was used as the vehicle for secretome delivery, and the biological response to the established biocomposite system was assessed by clinical, histological, histomorphometric, and microtomographic analysis. A comparative analysis revealed that the osteogenic-induced secretome presented an increased diversity of proteins, with emphasis on those related to osteogenesis. Microtomographic and histological morphometric analysis revealed that bioceramic xenografts implanted with secretomes enhanced the new bone formation process, with the osteogenic-induced secretome inducing the highest bone tissue formation. The application of the MSC secretome, particularly from osteogenic-induced populations, may be regarded as an effective therapeutic approach to enhance bone tissue healing and regeneration.

Survival rates from pancreatic cancer have remained stagnant for decades due to the heterogenic nature of the disease. This study aimed to find a new advanced biomarker and evaluate its clinical capabilities, thus enabling more individualised pancreatic cancer management. Between 2013 and 2020, 267 patients were included in the study. Surgically collected pancreatic tissue samples were analysed via high-definition mass spectrometry. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) was discovered as a possible promising pancreatic cancer biomarker. The predominance of CEACAM6 to pancreatic cancer was validated using antibodies in tissue samples. CEACAM6, carbohydrate antigen 19-9 (CA19-9), and carcinoembryonic antigen (CEA) blood serum concentrations were evaluated for clinical evaluation and comparison. Kaplan–Meier survival analyses were used to evaluate disease-free survival (DFS) and overall survival (OS). Poorer overall survival was significantly dependent on increased CEACAM6 blood serum concentrations (17.0 vs. 12.6 months, p = 0.017) in pancreatic cancer patients after radical treatment and adjuvant chemotherapy. Increased CEA and CA19-9 concentrations showed no significant dependencies with survival. Thus, CEACAM6 is a promising new biomarker with significant prognostic value and prediction of chemoresistance properties, enabling the improvement of individualised approaches to patients with pancreatic cancer.

Objectives Millions of people worldwide are affected by diseases or injuries which lead to bone/tooth loss and defects. While such clinical situations are daily practice in most of the hospitals, the widely used treatment methods still have disadvantages. Therefore, this field of medicine is actively searching new tissue regeneration techniques, one of which could be stem cell secretome. Thus, the purpose of this research study was to perform the detail proteomic analysis of periosteum-derived mesenchymal stem cells secretome in order to evaluate if it is capable to induce osteo-regenerative process. Material and Methods Periosteum-derived mesenchymal stem cells (PMSCs) were extracted from adult male New Zealand White rabbits. Cells were characterised by evaluating their differentiation potential. After characterisation PMSCs secretomes were collected and their proteomic analysis was performed. Results PMSCs were extracted from adult male New Zealand White rabbits. In order to characterise the extracted PMSCs, they were differentiated in the directions which mainly describes MSC multipotency - osteogenic, myogenic and adipogenic. A total of 146 proteins were detected. After characterisation PMSCs secretomes were collected and their proteomic analysis was performed. The resulting protein composition indicates the ability to promote bone regeneration to fully mature bone. Conclusions Bioactive molecules detected in periosteum-derived mesenchymal stem cells secretome initiates the processes required for the formation of a fully functional bone.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer due to the lack of diagnostic tools at the early stage and low efficiency of current chemotherapeutic approaches. The anticancer compounds with proven efficiency in established cell cultures often fail validation in further research. In this study, we employed PDAC patient-derived primary cell cultures to evaluate the efficiency of chemotherapeutic agents. Alongside, patients’ tissue samples were analysed by high-throughput differential proteomic analysis. We have shown that main firstline chemotherapeutic agents gemcitabine and FOLFIRINOX have little to no effect on the viability of patient-derived primary PDAC cells. The comparative proteomic and bioinformatic analysis of PDAC tumours shows an increase in the components of the extracellular matrix and focal adhesions and also overexpression of the downstream signaling from a variety of receptors, most notably PDGF receptor β and ErbB1 receptor. Consistently, all tumour-derived cell cultures assayed express a high level of PDGF receptor β. The enhancement of multiple signaling pathways leads to the increase in cell survival, proliferation, and resistance to apoptosis. Here we demonstrated the promising value of patient-derived primary PDAC cultures as a model for anticancer drug research and evaluation for individualized therapy.

The recombinant phage tail sheath protein, gp053, from Escherichia coli infecting myovirus vB_EcoM_FV3 (FV3) was able to self-assemble into long, ordered and extremely stable tubular structures (polysheaths) in the absence of other viral proteins. TEM observations revealed that those protein nanotubes varied in length (~10&ndash, 1000 nm). Meanwhile, the width of the polysheaths (~28 nm) corresponded to the width of the contracted tail sheath of phage FV3. The formed protein nanotubes could withstand various extreme treatments including heating up to 100 &deg, C and high concentrations of urea. To determine the shortest variant of gp053 capable of forming protein nanotubes, a set of N- or/and C-truncated as well as poly-His-tagged variants of gp053 were constructed. The TEM analysis of these mutants showed that up to 25 and 100 amino acid residues could be removed from the N and C termini, respectively, without disturbing the process of self-assembly. In addition, two to six copies of the gp053 encoding gene were fused into one open reading frame. All the constructed oligomers of gp053 self-assembled in vitro forming structures of different regularity. By using the modification of cysteines with biotin, the polysheaths were tested for exposed thiol groups. Polysheaths formed by the wild-type gp053 or its mutants possess physicochemical properties, which are very attractive for the construction of self-assembling nanostructures with potential applications in different fields of nanosciences.

Although treatment of colorectal cancer with 5-florouracil and oxaliplatin is widely used, it is frequently followed by a relapse. Therefore, there is an urgent need for profound understanding of chemotherapy resistance mechanisms as well as the profiling of predictive markers for individualized treatment. In this study, we identified the changes in 14 miRNAs in 5-fluouracil and 40 miRNAs in oxaliplatin-resistant cell lines by miRNA sequencing. The decrease in miR-224-5p expression in the 5-fluorouracil-resistant cells correlated with drug insensitivity due to its overexpression-induced drug-dependent apoptosis. On the other hand, the miR-23b/27b/24-1 cluster was overexpressed in oxaliplatin-resistant cells. The knockout of miR-23b led to the partial restoration of oxaliplatin susceptibility, showing the essential role of miR-23b in the development of drug resistance by this cluster. Proteomic analysis identified target genes of miR-23b and showed that endothelial–mesenchymal transition (EMT) was implicated in oxaliplatin insensibility. Data revealed that EMT markers, such as vimentin and SNAI2, were expressed moderately higher in the oxaliplatin-resistant cells and their expression increased further in the less drug-resistant cells, which had miR-23b knockout. This establishes that the balance of EMT contributes to the drug resistance, showing the importance of the miR-23b-mediated fine-tuning of EMT in oxaliplatin-resistant cancer cells.

When looking for the causes and treatments of infertility, much attention is paid to one of the reproductive tissues—the endometrium. Therefore, endometrial stem cells are an attractive target for infertility studies in women of unexplained origin. Menstrual blood stem cells (MenSCs) are morphologically and functionally similar to cells derived directly from the endometrium, with dual expression of mesenchymal and embryonic cell markers, they proliferate and regenerate better than bone marrow mesenchymal stem cells. In addition, menstrual blood stem cells are extracted in a non-invasive and painless manner. In our study, we analyzed the characteristics and the potential for decidualization of menstrual blood stem cells isolated from healthy volunteers and women diagnosed with infertility. We demonstrated that MenSCs express CD44, CD166, CD16, CD15, BMSC, CD56, CD13 and HLA-ABC surface markers, have proliferative properties, and after induction of menstrual stem cell differentiation into epithelial direction, expression of genes related to decidualization (PRL, ESR, IGFBP and FOXO1) and angiogenesis (HIF1, VEGFR2 and VEGFR3) increased. Additionally, the p53, p21, H3K27me3 and HyperAcH4 proteins’ expression increased during MenSCs decidualization, they secrete proteins that are involved in the regulation of the actin cytoskeleton, estrogen and relaxin signaling pathways and the management of inflammatory processes. Our findings reveal the potential use of MenSCs for the treatment of reproductive disorders.

BACKGROUND/AIM Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer, particularly due to its aggressive course and challenging diagnostics in early-stage disease. The aim of this study was to discover new potential prognostic and diagnostic pancreatic cancer biomarkers. MATERIALS AND METHODS The proteomes of 37 samples from pancreatic cancer, inflammatory or healthy pancreatic tissue derived through in-depth differential proteomic analysis were compared. RESULTS A set of candidate proteins as pancreatic cancer-specific diagnostic or prognostic biomarkers were identified. Survival data of patients after two-year follow-up indicated FLT3 and PCBP3 proteins as potential biomarkers for favourable pancreatic cancer prognosis. The levels of PCBP3 correlated with tumour stage and FLT3 levels, were evaluated as independent prognostic marker. CONCLUSION FLT3 and PCBP3 represent potential biomarkers for improved individualized pancreatic cancer prognosis. Moreover, FLT3 may play a role in future treatment selection.

A novel low-temperature Escherichia coli phage vB_EcoS_NBD2 was isolated in Lithuania from agricultural soil. With an optimum temperature for plating around 20 °C, vB_EcoS_NBD2 efficiently produced plaques on Escherichia coli NovaBlue (DE3) at a temperature range of 10–30 °C, yet failed to plate at temperatures above 35 °C. Phage vB_EcoS_NBD2 virions have a siphoviral morphology with an isometric head (65 nm in diameter), and a non-contractile flexible tail (170 nm). The 51,802-bp genome of vB_EcoS_NBD2 has a G + C content of 49.8%, and contains 87 probable protein-encoding genes as well as 1 gene for tRNASer. Comparative sequence analysis revealed that 22 vB_EcoS_NBD2 ORFs encode unique proteins that have no reliable identity to database entries. Based on homology to biologically defined proteins and/or proteomics analysis, 36 vB_EcoS_NBD2 ORFs were given a putative functional annotation, including 20 genes coding for morphogenesis-related proteins and 13 associated with DNA metabolism. Phylogenetic analysis revealed that vB_EcoS_NBD2 belongs to the subfamily Tunavirinae, but cannot be assigned to any genus currently recognized by ICTV.

Background and objective: Nitric oxide (NO) is known to exert cardioprotective effects against heart ischemic damage and may be involved in ischemic pre- and postconditioning. NO-triggered cardioprotective mechanisms are not well understood but may involve regulation of mitochondrial permeability transition pore (mPTP). In this study, we aimed to identify differentially phosphorylated mitochondrial proteins possibly involved in the NO/protein kinase G (PKG)/mPTP signaling pathway that can increase the resistance of cardiomyocytes to ischemic damage. Materials and methods: Isolated hearts from Wistar rats were perfused with NO donor NOC-18 prior to induction of stop&ndash, flow ischemia. To quantify and characterize the phosphoproteins, mitochondrial proteins were resolved and analyzed by two-dimensional gel electrophoresis followed by Pro-Q Diamond phosphoprotein gel staining, excision, trypsin digestions, and mass spectrometry. Quantitative proteomic analysis coupled with liquid chromatography&ndash, tandem mass spectrometry was also performed. Results: Mitochondrial protein phosphorylation patterns in NOC-18-pretreated ischemic hearts versus ischemic hearts were compared. Pretreatment of hearts with NOC-18 caused changes in mitochondrial phosphoproteome after ischemia which involved modifications of 10 mitochondrial membrane-bound and 10 matrix proteins. Among them, &alpha, subunit of ATP synthase and adenine nucleotide (ADP/ATP) translocase 1, both of which are considered as potential structural components of mPTP, were identified. We also found that treatment of isolated non-ischemic mitochondria with recombinant PKG did not cause the same protein phosphorylation as pretreatment of hearts with NOC-18. Conclusions: Our study suggests that pretreatment of hearts with NOC-18 causes changes in mitochondrial phosphoproteome after ischemia which involves modifications of certain proteins thought to be involved in the regulation of mPTP opening and intracellular redox state. These proteins may be potential targets for pharmacological preconditioning of the heart.

Peptidase family U32 is one of the few whose catalytic type and structure has not yet been described. It is generally accepted that U32 peptidases represent putative collagenases and contribute to the pathogenicity of some bacteria. Meanwhile, U32 peptidases are also found in nonpathogenic bacteria including thermophiles and hyperthermophiles. Here we report cloning of the U32.002 peptidase gene from thermophilic Geobacillus thermoleovorans DSM 15325 and demonstrate expression and characterization of the recombinant protein. It has been determined that U32.002 peptidase is constitutively expressed in the cells of thermophilic G. thermoleovorans DSM 15325. The recombinant oligomeric enzyme showed its activity only against heat-treated collagen. It was unable to degrade albumin, casein, elastin, gelatine and keratin. In contrast to this, the monomeric recombinant protein showed no activity at all. This paper is the first report about the thermophilic U32 peptidase. As the thermophilic bacteria are non-pathogenic, the role of constitutively expressed extracellular collagenolytic U32 peptidase in these bacteria is unclear.

Epigenetic changes play a significant role in leukaemia pathogenesis, therefore histone deacetylases (HDACis) are widely accepted as an attractive strategy for acute promyelocytic leukaemia (APL) treatment. Belinostat (Bel, PXD101), a hydroxamate-type HDACi, has proved to be a promising cure in clinical trials for solid tumours and haematological malignancies. However, insight into molecular effects of Bel on APL, is still lacking. In this study, we investigated the effect of Bel alone and in combination with differentiation inducer retinoic acid (RA) on human promyelocytic leukaemia NB4 and HL-60 cells. We found that treatment with Bel, depending on the dosage used, inhibits cell proliferation, whereas in combination with RA enhances and accelerates granulocytic leukaemia cell differentiation. We also evaluated the effect of used treatments with Bel and RA on certain epigenetic modifiers (HDAC1, HDAC2, PCAF) as well as cell cycle regulators (p27) gene expression and protein level modulation. We showed that Bel in combination with RA up-regulates basal histone H4 hyperacetylation level more strongly compared to Bel or RA alone. Furthermore, chromatin immunoprecipitation assay indicated that Bel induces the accumulation of hyperacetylated histone H4 at the p27 promoter region. Mass spectrometry analysis revealed that in control NB4 cells, hyperacetylated histone H4 is mainly found in association with proteins involved in DNA replication and transcription, whereas after Bel treatment it is found with proteins implicated in pro-apoptotic processes, in defence against oxidative stress and tumour suppression. Summarizing, our study provides some novel insights into the molecular mechanisms of HDACi Bel action on APL cells.

Bacteriophages represent a valuable source for studying the mechanisms underlying virus-host interactions. A better understanding of the host-specificity of viruses at the molecular level can promote various phage applications, including bacterial diagnostics, antimicrobial therapeutics, and improve methods in molecular biology. In this study, we describe the isolation and characterization of a novel coliphage, vB_EcoM_VpaE1, which has different host specificity than its relatives. Morphology studies, coupled with the results of genomic and proteomic analyses, indicate that vB_EcoM_VpaE1 belongs to the newly proposed genus Felix01likevirus in the family Myoviridae. The genus Felix01likevirus comprises a group of highly similar phages that infect O-antigen-expressing Salmonella and Escherichia coli (E. coli) strains. Phage vB_EcoM_VpaE1 differs from the rest of Felix01-like viruses, since it infects O-antigen-deficient E. coli strains with an incomplete core lipopolysaccharide (LPS). We show that vB_EcoM_VpaE1 can infect mutants of E. coli that contain various truncations in their LPS, and can even recognize LPS that is truncated down to the inner-core oligosaccharide, showing potential for the control of rough E. coli strains, which usually emerge as resistant mutants upon infection by O-Ag-specific phages. Furthermore, VpaE1 can replicate in a wide temperature range from 9 to 49 °C, suggesting that this virus is well adapted to harsh environmental conditions. Since the structural proteins of such phages tend to be rather robust, the receptor-recognizing proteins of VpaE1 are an attractive tool for application in glycan analysis, bacterial diagnostics and antimicrobial therapeutics.

Human amniotic fluid stem cells have become an attractive stem cell source for potential applications in regenerative medicine and tissue engineering. The aim of this study was to characterize amniotic fluid-derived mesenchymal stem cells (AF-MSCs) from second- and third-trimester of gestation. Using two-stage protocol, MSCs were successfully cultured and exhibited typical stem cell morphological, specific cell surface, and pluripotency markers characteristics. AF-MSCs differentiated into adipocytes, osteocytes, chondrocytes, myocytes, and neuronal cells, as determined by morphological changes, cell staining, and RT-qPCR showing the tissue-specific gene presence for differentiated cell lineages. Using SYNAPT G2 High Definition Mass Spectrometry technique approach, we performed for the first time the comparative proteomic analysis between undifferentiated AF-MSCs from late trimester of gestation and differentiated into myogenic, adipogenic, osteogenic, and neurogenic lineages. The analysis of the functional and expression patterns of 250 high abundance proteins selected from more than 1400 demonstrated the similar proteome of cultured and differentiated AF-MSCs but the unique changes in their expression profile during cell differentiation that may help the identification of key markers in differentiated cells. Our results provide evidence that human amniotic fluid of second- and third-trimester contains stem cells with multilineage potential and may be attractive source for clinical applications.

Uncontrolled proliferation is the hallmark of cancer and other proliferative disorders and abnormal cell cycle regulation is, therefore, common in these diseases. Cyclin-dependent kinases (CDKs) play a crucial role in the control of the cell cycle and proliferation. These kinases are frequently deregulated in various cancers, viral infections, neurodegenerative diseases, ischemia and some proliferative disorders. This led to a rigorous pursuit for small-molecule CDK inhibitors for therapeutic uses. Early efforts to block CDKs with nonselective CDK inhibitors led to little specificity and efficacy but apparent toxicity, but the recent advance of selective CDK inhibitors allowed the first successful efforts to target these kinases for the therapies of several diseases. Major ongoing efforts are to develop CDK inhibitors as monotherapies and rational combinations with chemotherapy and other targeted drugs.

The name of a funding organization is given incorrectly in the Funding Statement. The Funding Statement should read: "This work was funded by the Research Council of Lithuania (SVE-04/2012). URL: http://www.lmt.lt/."

At 346 kbp in size, the genome of a jumbo bacteriophage vB_KleM-RaK2 (RaK2) is the largest Klebsiella infecting myovirus genome sequenced to date. In total, 272 out of 534 RaK2 ORFs lack detectable database homologues. Based on the similarity to biologically defined proteins and/or MS/MS analysis, 117 of RaK2 ORFs were given a functional annotation, including 28 RaK2 ORFs coding for structural proteins that have no reliable homologues to annotated structural proteins in other organisms. The electron micrographs revealed elaborate spike-like structures on the tail fibers of Rak2, suggesting that this phage is an atypical myovirus. While head and tail proteins of RaK2 are mostly myoviridae-related, the bioinformatics analysis indicate that tail fibers/spikes of this phage are formed from podovirus-like peptides predominantly. Overall, these results provide evidence that bacteriophage RaK2 differs profoundly from previously studied viruses of the Myoviridae family.